DESCRIPTION (Investigator's Abstract): The study of the basic mechanisms involved in the regulation of corneal epithelial metabolism by basal lamina components is important to the understanding of normal development of corneal epithelia, cell migration during wound repair, and basement membrane diseases such as recurrent corneal erosions and corneal dystrophies. The long-term objective of this grant is to determine the functional and structural relationships between the basement membrane, corneal epithelial cytoskeleton, and organelle distribution (rough endoplasmic reticulum, Golgi). We will be testing the hypothesis that the basal lamina and extracellular matrix proteins determine cytoskeletal organization via surface receptors. In turn the cytoskeleton changes rough endoplasmic reticulum organization or other transcriptional and translational factors that regulate protein synthesis. I have proposed three specific aims that will test this hypothesis. 1) Does the actin cytoskeleton organize other cytoskeletal proteins and organelles involved in secreted protein synthesis? 2) Is the extracellular matrix-stimulated increase in collagen synthesis regulated by transcriptional, translational, or post-translational mechanism? Are co-translational factors or rough endoplasmic reticulum stabilized by attachment to cytoskeleton? 3) What is the spatial distribution of all secreted protein organelles (rough endoplasmic reticulum, Golgi, and secretory vesicles) and is their distribution cytoskeletal dependent? What is the distribution of specific mRNA for secreted proteins (collage)? Are the specific collagen mRNA distributions cytoskeletal dependent? Recently, I've developed techniques to study epithelial tissues with confocal microscopy. The development of these techniques allows us to determine the spatial relationships between specific cellular components, such as the cytoskeletal proteins and rough endoplasmic reticulum or specific mRNA. In the future, I hope to develop techniques to visualize these interactions in the living intact corneal epithelial tissue. In summary, a cell biological model for corneal epithelia interactions between the extracellular environment and cellular organization has been developed. New techniques will be combined with classical immunohistochemistry, biochemistry, and molecular biology to test the hypothesis that cytoskeletal elements organize protein secretion organelles and translation cofactors to regulate expression of secreted proteins.